© 2019, Association of Food Scientists & Technologists (India).Enzyme resistant starch (ERS) is a indigestible form of starch and it can behave like dietary fibre that are the indigestible food components essential for a healthy lifestyle. The aim of this study was to examine the changes of the ERS content of wheat flour with ultrasonication, at four different flour/water ratios (1/5, 1/10, 1/15 and 1/20), three different pH values (6.1, 5.5 and 4.5) and three different temperatures (80 °C, 100 °C and 121 °C). Furthermore, possibility of addition this flour in four different proportions (0%-control, 25%, 50% and 75%) for biscuit production as a dietary fibre source was also investigated. The applied processes increased the dark colour and water holding capacity, but decreased the moisture content of flour samples. The ERS content of wheat flour increased about 30-fold and the sample that had the highest ERS content (ERS-enriched flour) of 3.30% was produced at 121 °C (autoclaving), pH 6.1 and 1/15 flour/water ratio with ultrasonication. ERS-enriched flour had higher melting point and lower estimated glycemic index (eGI) value compared to the wheat flour. In biscuit production, the biscuits with medium eGI (61.82–67.31) was obtained and the increase of ERS-enriched flour proportion increased the ERS content of biscuits, decreased the eGI, hardness and fracturability. During storage, while the light colour and moisture content of biscuits increased; eGI, hardness and fracturability decreased. The biscuits were considered as sensory acceptable by panellists. As conclusion; the ERS-enriched flour can contribute to the production of the medium or low GI foods rich in carbohydrates.